HBV (Hepatitis B virus) is a major cause of acute and chronic hepatitis worldwide. Prevention of HBV infection may be achieved with active or passive immunization. Active immunization with recombinant HBV vaccines can prevent HBV infection if given before exposure. Despite the introduction of universal vaccination against hepatitis B in around 100 countries, persistent HBV infection is still a serious problem, with an estimated worldwide death rate of one million people per year. Protective antibodies that appear after natural infection are mostly directed against the major antigenic ‘a’ determinant of hepatitis B surface antigen (HBsAg). The immunodominant ‘a’ epitope is present in all serotypes. Antibodies against HBsAg are thus advocated for passive immunotherapy for managing chronic viral infection. Passive immunization with hepatitis B specific antibodies, given shortly after exposure, can decrease the incidence or severity of disease.
Currently, Hepatitis B immune globulin (HBIG), collected from the blood of hyper immune human donors, is used for the post-exposure prophylaxis in cases of accidental needle stick injuries, liver transplant patients and for the prevention of vertical transfer of HBV infection form mother to child. This blood-derived product is not manufactured in India. However, such blood-derived products are costly and can cause cross contamination. Therefore, recombinant antibodies can be good substitutes for human serum antibodies.
Inventors developed a mouse monoclonal antibody (5S) against the ‘a’ epitope of the hepatitis B surface antigen (HBsAg). However such mouse antibodies often induce a significant human anti mouse antibody (HAMA) response when administered to patients and thus limiting their potential use for human therapy, especially when repeated administration is necessary. HAMA greatly reduces the in-vivo efficacy of mouse antibodies. Moreover, the half-life of a mouse antibody in human plasma is shorter compared with that of human antibody. Several recombinant anti-HbsAg antibody fragments have been reported in literature.
However most of them are of mouse origin and are not available for therapeutic purposes (1-15). To reduce the immunogenicity of murine antibodies, chimeric antibodies were constructed, which combine the variable region of a mouse antibody with a human antibody constant region, thus retaining the binding specificity of murine antibody while presenting less foreign amino acid sequence to the human immune system (16). Inventors also generated chimeric antibody against HBsAg (17-18). Some of the chimeric antibodies have proved less immunogenic in humans, whereas others are almost as immunogenic as murine antibodies. Moreover, in an animal study to evaluate the immunogenicity of chimeric antibodies, it was found that the anti-variable domain response was not attenuated in the chimeric antibodies, suggesting that the murine variable domain can still lead to a significant human anti mouse antibody (HAMA) response (19). Therefore, for therapeutic purposes it may be necessary to fully humanize a murine antibody by altering the variable domain to make them human like. It is well established that humanization of mouse antibody is desirable to reduce its potential product immunogenicity. However humanization is practical only if it does not reduce or destroy the binding affinity of antibody. Humanized antibodies are safer for therapeutic uses and currently several such humanized antibodies are in clinical uses for various diseases. Although some chimeric antibodies are in clinical use, it is worth noting that most of the antibodies in phase I, II and III clinical trials today are humanized antibodies.
All the mouse/humanized and human anti-HbsAg antibodies reported in literature have unique complementarily determining regions (CDRs) sequence and have unique antigen-antibody interactions which are different form the recombinant molecule of this invention.    1. Ehrlich P H, Moustafa Z A, Justice J C. Harfeldt K E, Kelley R L, Osterg L. Characterization of human monoclonal antibodies directed against hepatitis B surface antigen. Hum Antibodies Hybridomas. 1992 January; 3(1):2-7.    2. Kennedy R C, lonescu-Matiu I, Adler-Storthz K, Henkel R D Sanchez Y, Dreesman G R. Characterization of anti-hepatitis B surface antigen monoclonal antibodies. Intervirology. 1983; 19(3): 176-80.    3. Heijtink R A, Kruining J, Weber Y A, de Man R A. Schaim S W. Anti-hepatitis B virus activity of a mixture of two monoclonal antibodies in an “inhibition in solution” assay. Hepatology. 1995 October; 22(4 Pt 1): 1078-83.    4. Shouval D, Wands J R, Zurawski V R Jr. Isselbacher K J, Shafritz D A. Selecting binding and complement-mediated lysis of human hepatoma cells (PLC/PRF/5) in culture by monoclonal antibodies to hepatitis B surface antigen. Proc Natl Acad Sci USA. 1982 January; 79 (2): 650-4.    5. Shin Y W, Ryoo K H, Hong K W, Chang K H, Choi J S, So M, Kim P K, Park J Y, Bong K T, Kim S H. Human monoclonal antibody against Hepatitis B virus surface antigen (HbsAg). Antiviral Res. 2007 August; 75 (2): 113-20.    6. Wands J R. Zurawski V R Jr. High affinity monoclonal antibodies to hepatitis B surface antigen (HbsAg) produced by somatic cell hybrids. Gastroenterology. 1981 February; 80(2): 225-32.    7. Falero G. RodrAguez l, Sarracent J. Otero A J, RodrAguez B L, Rojas A, Ochoa E. Generation of murine triomas secreting bi-specific monoclonal antibodies that recognize HbsAG ad and ay subtypes. Hybridoma. 1992 December; 11 (6):815-23.    8. Shearer M H, Sureau C, Dunbar B, Kennedy R C. Structural characterization of viral neutralizing monoclonal antibodies to hepatitis B surface antigen. Mol immunol. 1998 December; 35(18):1149-60.    9. Maeda F, Nagatsuka Y, lhara S, Aotsuka S, Ono Y, Inoko H, Takekoshi M. Bacterial expression of a human recombinant monoclonal antibody fab fragment against hepatitis B surface antigen. J Med. Virol. 1999 August; 58(4):338-45.    10. Lohman K L, Kieber-Emmons T, Kennedy R C. Molecular characterization and structural modeling of immunoglobulin variable regions form murine monoclonal antibodies specific for hepatitis B virus surface antigen. Mol. Immunol. 1993 October; 30(14):1295-306.    11. Heijtink R A, Kruining J, van Bergen P, de Rave S, van Hattum J. Schutten M, Osterhaus A D. Characterization of a human monoclonal antibody obtained after immunization with plasma vaccine and a booster with recombinant-DNA hepatitis B vaccine. J Med. Virol. 2002 March; 66(3):304-11.    12. Yano A, Maeda F, Takekoshi M. Transgenic tobacco cells producing the human monoclonal antibody to hepatitis B virus surface antigen. J Med. Virol. 2004 June; 73(2):208-15.    13. Desgranges C. Paire J, Pichoud C, Souche S, Frommel D, Trepo C. High affinity human monoclonal antibodies directed against hepatitis B surface antigen. J Virol Methods. 1987 July; 16 (4):281-92.    14. Ryu C J, Padlan E A, Jin B R, Yoo O J, Hong H J. A humanized antibody with specificity for hepatitis B surface antigen. Hum Antibodies Hybridomas. 1996; 7(3):113-22.    15. Eren R. Lubin l, Terkieltaub D, Ben-Moshe O, Zauberman A, Uhlmann R, Tzahor T, Moss S, llan E, Shouval D, Galun E, Daudi N, Marcus H, Reisner Y, Dagan S. Human monoclonal antibodies specific to hepatitis B virus generated in a human/mouse radiation chimera: the Trimera system. Immunology. 1998 February; 93(2):154-612.    16. Morrison S L, Johnson M J, Herzenberg L A, Oi V T. Chimeric human antibody molecules; mouse antigen-binding domains with human constant region domains. Proc Natl Acad Sci USA. 1984 November; 81(21):6851-5.    17. Bose B, Khanna N, Acharys S K, Sinha S. High affinity mouse—human chimeric Feb against hepatitis B surface antigen. World J Gastroenterol. 2005 Dec. 28; 11(48); 7569-78.    18. Bose B, Khanna N, Acharya S K, Sinha S. Generation and characterization of a high-affinity chimeric antibody against hepatitis B surface antigen. Biotechnol Appl Biochem. 2006 February; 43(Pt2):93-101.    19. Khazaeli M B, Saleh M N, Liu T P, Meredith R F, Wheeler R H, Baker T S, King Secher D, Allen L, Rogers K, et al. Pharmacokinetics and immune response of 131l-chimeric mouse/human B72.3 (human gamma 4) monoclonal antibody in humans. Cancer Res. 1991 Oct. 15; 51 (20): 5461-6